JP4640742B2 - Method and means for producing a high-titer and safe recombinant lentiviral vector - Google Patents
Method and means for producing a high-titer and safe recombinant lentiviral vector Download PDFInfo
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- JP4640742B2 JP4640742B2 JP2000539150A JP2000539150A JP4640742B2 JP 4640742 B2 JP4640742 B2 JP 4640742B2 JP 2000539150 A JP2000539150 A JP 2000539150A JP 2000539150 A JP2000539150 A JP 2000539150A JP 4640742 B2 JP4640742 B2 JP 4640742B2
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Description
【0001】
発明の分野
本発明は、新規レンチウイルスパッケージングベクター、問題の外来遺伝子を保持する導入ベクター、安定したパッケージング細胞株、安定したプロデューサー細胞株、及び哺乳類細胞中で組換えレンチウイルスを産生するためのそれらの使用に関する。
発明の背景
レトロウイルスベクターは、遺伝子送達のための一般的道具である(Miller 、 Nature 、357:455-460(1992))。レトロウイルスベクターの再編成されない単一コピー遺伝子を広い範囲の齧歯類、霊長類及びヒトの体細胞に送達する能力は、細胞への遺伝子導入に良く適したレトロウイルスベクターを作出する。
【0002】
組換えレトロウイルスベクターを作製するために有用な付属物は、感染性ビリオンの作製に必要なタンパク質をトランス(in trans)で供給するパッケージング細胞株であるが、このような細胞は、内因性のウイルスゲノム核酸をパッケージングすることが不可能である(Watanabe 及びTemin 、Molec. Cell Biol. 、3(12):2241-2249(1983) ;Mannら、Cell、33:153-159(1983);Embretson 及びTemin 、J. Viol.、61(9):2675-2683(1987) ) 。レトロウイスルパッケージング細胞株の構築に関する考察は、齧歯類(Cloydら、J. Exp. Med.、151:542-552(1980))及び霊長類(Donahueら、J. Exp. Med.、176:1125-1135(1992))においてTリンパ球を産生することが示されている、組換えの複製コンピテントのレトロウイルス(RCR) を含まない高力価のベクターの上清の作製である。
【0003】
パッケージング細胞においてRCR の生成の可能性を最小にするひとつの方法は、パッケージング機能を2個のゲノム、例えば、一方はgag 及びpol 遺伝子産物を発現し、他方はenv 遺伝子産物を発現するものに分割することである(Bosselmanら、Molec. Cell Biol. 、7(5):1797-1806(1987);Markowitz ら、J. Virol. 、62(4):1120-1124(1988) ;Danos 及びMulligan、Proc. Natl. Acad. Sci.、85:6460-6464(1988))。この方法は、これらの2個のゲノムの同時パッケージング及びそれに続く導入の能力、更にはRCR を産生するための該パッケージング細胞における3個のレトロウイルスゲノムの存在に起因した組換え頻度を顕著に減少する能力を最小化する。
【0004】
組換え体が生じる事象において、いずれか可能性のある組換え体を機能しなくするために望ましくない遺伝子産物内に変異(Danos及びMulligan、前記)又は欠失(Boselman ら、前記;Markowitz ら、前記)を設計することができる。加えて、両パッケージング構築物上の3’側LTR の欠失は、機能的組換え体を形成する能力も低下させる。
【0005】
レンチウイルスは、一般的なレトロウイルス遺伝子gag 、pol 及びenv に加えて、調節又は構造機能を伴う他の遺伝子を含む複合レトロウイルスである。高度な複雑性は、潜伏感染期に、レンチウイルスがその生活環を変調することを可能にする。
典型的レンチウイルスは、AIDSの病原物質であるヒト免疫不全ウイルス(HIV) である。In vivo においてHIV は、リンパ球及びマクロファージのような分裂が稀である最終的に分化した細胞に感染することができる。In vitroにおいて、HIV は、単球由来マクロファージ(MDM) の初代培養、更にはアフィディコリン又はγ線照射の処理によって細胞周期が抑制されたHeLa-Cd4細胞又はTリンパ球細胞に感染することができる。
【0006】
細胞の感染は、標的細胞の核膜孔を通じてのHIV の組込み前複合体の核への能動輸送に左右される。これは、標的細胞の核への輸送機構と、該複合体中の複数の部分的に多重複の分子決定基の相互作用によって生じる。同定された決定基は、gag マトリックス(MA)タンパク質、親核性の(karyophilic)ビリオン−関連タンパク質、vpr 、及びgag MAタンパク質のC-末端ホスホチロシン残基の機能的核局在化シグナル(NLS)を含む。
発明の概要
従って、本発明は、パッケージできるレンチウイルスベクター転写物及び哺乳類細胞における高力価組換えレンチウイルスの迅速な作製のためのレンチウイルスタンパク質の両方の合成に関係する新規の安全化されたレンチウイルスベクターに関する。これらの結果は、問題の外来遺伝子を標的細胞へ送達するための感染性粒子である。本発明は更に、ウイルス産生のための細胞株を提供する。
発明の詳細な説明
本発明は、非分裂細胞を感染することが可能な組換えレンチウイルス、更にはこれを製造する方法及び手段を提供する。このウイルスは、核酸配列のin vivo 及びex vivo における導入及び発現に有用である。
【0007】
レンチウイルスゲノム及びプロウイルスDNA は、レトロウイスルにおいて発見された3個の遺伝子を有する:gag 、pol 及びenv であり、これらには、2個の長い末端反復配列(LTR) が隣接している。gag 遺伝子は、内部構造(マトリックス、キャプシド及びヌクレオキャプシド)タンパク質をコードし;pol 遺伝子は、RNA-依存性DNA ポリメラーゼ(逆転写酵素)、プロテアーゼ及びインテグラーゼをコードし;並びに、env 遺伝子は、ウイルスのエンベロープの糖タンパク質をコードしている。5’及び3’側LTR は、ビリオンRNA の転写及びポリアデニル化を促進するのに利用できる。LTR は、ウイルス複製に必要な他のシス作用性配列を全て含む。レンチウイルスは、vif 、vpr 、tat 、rev 、vpu 、nef 及びvpx (HIV-1 、HIV-2 及び/又はSIV )を含む追加遺伝子を有する。
【0008】
5’側LTR への隣接物は、ゲノムの逆転写に必要な配列(tRNAプライマー結合部位)及びウイルスRNA の粒子への効果的なキャプシド形成に必要な配列(プサイ部位)である。キャプシド形成(又はレトロウイルスRNA の感染ビリオンへのパッケージング)に必要な配列がウイルスゲノムから失われた場合は、このシス欠損は、ゲノムRNA のキャプシド形成を妨げる。しかし、得られる変異体は、依然全てのビリオンタンパク質の合成を指向することが可能である。
【0009】
本発明は、パッケージング機能、すなわちgag 、pol 及びenv 、更にはrev 及びtat を有する2種以上のベクターにより適当な宿主細胞へトランスフェクションすることを含む、非分裂細胞を感染することが可能な組換えレンチウイルスを作製する方法を提供する。以下に説明するように、機能性tat 遺伝子を欠いているベクターが、特定の用途のためには望ましい。従って、パッケージング細胞を産生するために、例えば第一のベクターは、ウイルス性gag 及びウイルス性pol をコードしている核酸を提供することができ、かつ別のベクターは、ウイルス性env をコードしている核酸を提供することができる。本願明細書において導入ベクターと同定される異種遺伝子を提供するベクターのパッケージング細胞への組入れは、問題の外来遺伝子を保持する感染性ウイルス粒子を放出するプロデューサー細胞を生じる。
【0010】
前述のベクターそれ自体は、本願明細書において開示された新規に構築されたベクターの範囲外であるが、これは当該技術分野において公知であり、Naldini らの論文(Sci.、272:263-267(1996) );及びZuffereyらの論文(Nat. Biotech. 、15:871-875(1997))を参照のこと。一般にこれらのベクターは、プラスミドをベースにした又はウイルスをベースにしたものであり、外来核酸の組入れ、核酸の選択及び宿主細胞への導入に必須の配列を保持するように設計されている。問題のベクターのgag 、pol 及びenv 遺伝子も、当該技術分野において公知である。従って関連する遺伝子が、選択されたベクターにクローニングされ、その後問題の標的細胞の形質転換に使用される。
【0011】
前述のベクター及び外来遺伝子の設計に従い、第二のベクターは、ウイルス性エンベロープ(env) 遺伝子をコードしている核酸を提供することができる。env 遺伝子は、レトロウイルスを含むあらゆるウイルスに由来することができる。好ましくはenv は、ヒト及び他種の細胞の形質導入を可能にする両種指向性エンベロープタンパク質である。
【0012】
このエンベロープタンパク質の抗体との結合、又は特定の細胞種のレセプターへの標的化のための特定のリガンドにより組換えウイルスを標的化することが望ましい。ウイルスベクターに問題の配列(調節領域を含む)を、例えば特異的標的細胞上の受容体のリガンドをコードしている他の遺伝子と共に挿入することにより、このベクターは標的特異性となる。レトロウイルスベクターは、例えば糖脂質又はタンパク質の挿入により、標的特異性に作製することができる。標的化は、抗体又は組換え抗体型分子、例えば1本鎖抗体などの抗原−結合部分を使用することにより達成され、レトロウイルスベクターを標的化する事が多い。当業者には、特異的標的へのレトロウイルスベクターの送達を達成する具体的な方法は公知であるか、又は過度の実験をすることなく容易に確かめるであろう。
【0013】
レトロウイルス−由来のenv 遺伝子の例は、モロニーマウス白血病ウイルス(MoMuLV 又はMMLV) 、ハーベイマウス肉腫ウイルス(HaMuSV又はHSV )、マウス乳がんウイルス(MuMTV 又はMMTV)、ギボンサル白血病ウイルス(GaLV又はGALV)、ヒト免疫不全ウイルス(HIV) 及びラウス肉腫ウイルス(RSV) を含むが、これらに限定されるものではない。水泡性口内炎ウイルス(VSV) プロテインG(VSVG) 、肝炎ウイルス及びインフルエンザのそれのような他のenv 遺伝子も使用することができる。
【0014】
ウイルス性env 核酸配列を提供するベクターは、例えばプロモーター又はエンハンサーのような調節配列に機能的に結合している。この調節配列は、真核生物のプロモーターまたはエンハンサーであることができ、例えばモロニーマウス白血病ウイルスのプロモーター−エンハンサーエレメント、ヒトサイトメガロウイルスのエンハンサー又はワクシニアP7.5のプロモーターを含む。一部の場合は、モロニーマウス白血病ウイルスのプロモーター−エンハンサーエレメントのように、プロモーター−エンハンサーエレメントが、LTR 配列内に又はこれに隣接して位置する。
【0015】
好ましくはこの調節配列は、ベクターが構築されたレンチウイルスにとって内因性でないものである。従って、ベクターがSIV から作出された場合は、SIV LTR に認められるSIV 調節配列は、SIV を起源としない調節エレメントによって置き換えられるであろう。
VSV G は組換えウイルスに広範な宿主細胞の範囲を付与するので、VSV G タンパク質は望ましいenv 遺伝子であるが、VSV G は宿主細胞にとって有害であり得る。従って、VSV G に関する遺伝子のような遺伝子が使用される場合は、誘導性プロモーターシステムを使用し、その結果VSV G 発現が必要でない場合には、VSV G 発現を宿主毒性が最小になるように調節できるようにすることが好ましい。
【0016】
例えばGossen及びBujardの論文(Proc. Natl. Acad. Sci.、89:5547-5551(1992))に記載されたテトラサイクリン−調節性遺伝子発現システムを用いて、テトラサイクリンが導入された細胞から取り除かれた場合にVSV G の誘導発現を提供するために利用することができる。従って、tet/VP16トランスアクチベーターは、第一のベクター上に存在し、かつVSV G をコードする配列は、別のベクター上のtet オペレーター配列によって制御されたプロモーターの下流にクローニングされる。
【0017】
異種又は外来の核酸配列である導入遺伝子は、調節核酸配列に操作できるように連結される。本願明細書において使用される用語「異種」核酸配列は、外来種を起源とする配列、又は同種に由来する場合にはこれが当初の形から実質的に修飾されていることを意味する。あるいは、細胞では通常は発現しない未変化の核酸配列は、異種核酸配列である。
【0018】
用語「操作できるように連結」とは、調節配列と異種核酸配列が機能的に連結し、後者の発現を生じることを意味する。好ましくは、異種配列は、プロモーターに連結され、キメラ遺伝子を生じる。この異種核酸配列は、好ましくはウイルス性LTR プロモーター−エンハンサーシグナル又は内部プロモーターのいずれかの制御下にあり、レトロウイスルLTR 内に保持されたシグナルは、依然導入遺伝子の効果的発現をもたらすことができる。
【0019】
前記外来遺伝子は、転写することができる問題の核酸のいずれかであることができる。一般に外来遺伝子はポリペプチドをコードしている。このポリペプチドは、何らかの治療上の利点があるものが好ましい。ポリペプチドは、宿主細胞において、内因性タンパク質の発現の欠損又は不存在を補うことができる。このポリペプチドは、宿主細胞に新たな特性、例えば米国特許第5,359,046 号に開示されたキメラシグナル受容体などを与えることができる。技術者は、本願明細書に記載しかつ当該技術分野において公知である技術を実践し外来遺伝子の適性を決定することができる。例えば、技術者は、外来遺伝子はキャプシド形成に適した大きさかどうか、及び外来遺伝子産物が適切に発現されるかどうかということを知っているであろう。
【0020】
本発明の方法による分子の組入れにより細胞において分子を調節する遺伝子の発現を変調することが望ましい。用語「変調」は、過剰発現された場合の遺伝子発現の抑制、又は過小発現された場合の発現の増大を描いている。細胞の増殖障害が遺伝子発現に関連している場合は、翻訳レベルで遺伝子発現を妨害している核酸配列を使用することができる。この方法は、例えばアンチセンス核酸リボザイム又はトリプレックス物質(triplex agent) を用いて、核酸又はトリプレックス物質によりmRNAを隠すか、又はリボザイムでこれを切断するかのいずれかにより、特異的mRNAの転写又は翻訳を阻害することができる。
【0021】
アンチセンス核酸は、特異的mRNA分子の少なくとも一部と相補的であるDNA 又はRNA 分子である(Weintraub、Sci. Am.、262:40(1990)) 。細胞において、アンチセンス核酸は、対応するmRNAにハイブリダズし、2本鎖分子を形成する。細胞は2本鎖mRNAを翻訳しないので、アンチセンス核酸はmRNAの翻訳を妨害する。約15個以上のヌクレオチドのアンチセンスオリゴマーが、容易に合成され、かつ恐らく標的細胞に組入れられた際により大きい分子よりも問題を引き起こすことが少ないので、好ましい。遺伝子のin vitro翻訳を阻害するためのアンチセンス法の使用は、当該技術分野において周知である(Marcus-Sakura 、Anal. Biochem.、172:289(1988))。
【0022】
アンチセンス核酸は、アルツハイマー病において蓄積するアミロイド前駆体タンパク質のような変異タンパク質又はドミナントアクティブ遺伝子産物の発現をブロックするために使用することができる。このような方法は、更にハンチントン病、遺伝性パーキンソン病及び他の疾患の治療についても有用である。アンチセンス核酸は、毒性に関連したタンパク質の発現の阻害についても有用である。
【0023】
転写を停止するためのオリゴヌクレオチドの使用は、オリゴマーが二重らせんDNA の周りに巻きつき、3本鎖らせんを形成するトリプレックス戦略として公知のメカニズムによることができる。従って、トリプレックス化合物は、選択された遺伝子上の独自の部位を認識するように設計することができる(Maher ら、Antisense REs and Dev.、1(3)227(1991) ;Helene、Anticancer Drug Dis.、6(6):569(1991))。
【0024】
リボザイムとは、他の1本鎖RNA をDNA 制限エンドヌクレアーゼに類似の方法で特異的に切断する能力を有するRNA 分子である。これらのRNA をコードしているヌクレオチド配列の修飾を通じて、RNA 分子において特異的ヌクレオチド配列を認識し切断する分子を操作することが可能である(Cech、J. Amer. Med. Assn. 、260:3030(1988))。このような方法の大きい利点は、特定の配列を有するmRNAのみが不活性化されることである。
【0025】
生体反応調節剤をコードしている核酸を導入することが望ましい。この範疇に含まれるのは、「インターロイキン」に分類される多くのサイトカイン類、例えばインターロイキン1から12をコードしている核酸を含む免疫賦活剤である。更にこの範疇に含まれるのは、必ずしも同じメカニズムに従って作用するものではないが、インターフェロン、特にγインターフェロン(γ-IFN) であり、腫瘍壊死因子(TNF) 及び顆粒球−マクロファージコロニー刺激因子(GM-CSF)である。このような核酸が骨髄細胞又はマクロファージに送達され、先天性の酵素欠損又は免疫不全を治療することが望ましい。増殖因子、毒素性ペプチド、リガンド、受容体又は他の生理学的に重要なタンパク質をコードしている核酸も、特異的非分裂細胞に組入れることができる。
【0026】
従って、本発明の組換えレンチウイルスは、抗HIV-分子によりHIV-感染細胞(例えばT細胞又はマクロファージ)を治療するために使用することができる。加えて、例えば、気道上皮を、嚢胞性繊維症の治療のために、嚢胞性繊維症トランスメンブランコンダクタンス調節(CFTR)遺伝子を有する本発明の組換えレンチウイルスにより感染することができる。
【0027】
本発明の方法は、更にex vivo での本発明の組換えレンチウイルスに感染した細胞の移植、又は中枢神経系もしくは脳室洞(ventricular cavity)への又は宿主脳の表面の硬膜下へのin vivo での感染に関連している、神経細胞、グリア細胞、繊維芽細胞又は間充織細胞の移植(transplantation) 又は「グラフト」に有用である。このようなグラフト化に関する方法は、当業者には周知であり、かつBjorklund 及びStenevi 編集のNeural Grafting in the Mammalian CNS(1985年)に記載されている。
【0028】
タンパク質産物の欠損に起因する疾患に関して、遺伝子導入は、代償療法のために正常遺伝子を感染組織へ組入れることができ、更にはアンチセンス変異を用いた疾患のための動物モデルを作製することができる。例えば、筋肉、脾臓又は肝臓細胞の感染のためには、ファクターVIII又はIXをコードする核酸をレンチウイルスに挿入することが望ましい。
【0029】
プロモーター配列は、所望の遺伝子配列に対して同種又は異種であることができる。広範なプロモーター類を利用することができ、これはウイルス又は哺乳類のプロモーターを含む。細胞又は組織に特異的なプロモーターは、特異的細胞集団における遺伝子配列の発現の標的指向化に利用することができる。本発明に適した哺乳類又はウイルスプロモーターは、当該技術分野において入手することができる。
【0030】
任意に、クローニング段階の間に、パッケージングシグナル及び異種クローニング部位を有する導入ベクターと称される核酸構築物は、更に選択マーカー遺伝子を含む。マーカー遺伝子は、該ベクターの存在を調べるために利用され、その結果感染又は組込みが確認される。マーカー遺伝子の存在は、該挿入物を発現している宿主細胞のみの選択及び増殖を保証する。典型的な選択遺伝子は、抗生物質又は他の毒物、例えばヒスチジノール、プロマイシン、ハイグロマイシン、ネオマイシン、メトトレキセートなどに対する耐性を付与するタンパク質、及び細胞表面マーカーをコードしている。
【0031】
本発明の組換えウイルスは、哺乳類細胞に核酸配列を導入することが可能である。用語「核酸配列」は、本願明細書において詳細に論ぜられるように、いずれかの核酸分子、特にDNA を意味する。核酸分子は、DNA 、cDNA、合成DNA 、RNA 又はそれらの組合せを含む様々な起源に由来することができる。このような核酸配列は、天然のイントロンを含むこともあり、含まないこともあるようなゲノムDNA を包含し得る。更にこのようなゲノムDNA は、プロモーター領域、ポリA配列又は他の関連する配列に関連して得ることができる。ゲノムDNA は、当該技術分野において周知の手段により、適当な細胞から抽出及び精製することができる。あるいは、メッセンジャーRNA(mRNA) は、細胞から単離し、逆転写又は他の手段によりcDNAを作製するために使用することができる。
【0032】
好ましくは、本発明の方法で作製された組換えレンチウイルスは、ヒト免疫不全ウイルス(HIV) の誘導体である。このenv はHIV 以外のウイルスに由来するであろう。
本発明の方法は、いくつかの実施態様において、前述のように、例えばgag 、pol 、env 、tat 及びrev のような組換えビリオンのパッケージングに必要な全ての機能をもたらす3種のベクターを提供する。本願明細書において記したように、tat は、予想外の利点を機能的に欠失し得る。これらのベクターがパッケージング細胞株の形質転換及び作出に使用され、組換えレンチウイルスを生じる限りは、使用されるベクターの数に制約はない。
【0033】
前述のベクターは、トランスフェクション又は感染によりパッケージング細胞株に組入れられる。このパッケージング細胞株は、該ベクターゲノムを含むウイルス粒子を産生する。トランスフェクション又は感染の方法は、当業者には周知である。パッケージングベクター及び導入ベクターのパッケージング細胞株への同時トランスフェクション後、当業者によって用いられる常法により組換えウイルスは培養培地から回収され、かつ力価測定される。
【0034】
従って、このパッケージング構築物は、リン酸カルシウムトランスフェクション法、リポフェクション法又は電気穿孔法により、通常はneo 、DHFR、Gln シンテターゼ又はADA のようなドミナントな選択マーカーと一緒に、ヒト細胞株に導入し、その後適当な薬物の存在下で選択しかつクローンを単離することができる。これらの選択マーカー遺伝子は、該構築物の中のパッケージング遺伝子に物理的に連結することができる。
【0035】
パッケージング機能が適当なパッケージング細胞によって発現されるように設計された安定した細胞株は公知である。例えばパッケージング細胞について開示した米国特許第5,686,279 号;及びにOrayらの論文(Proc. Natl. Acad. Sci.、93:11400-11406(1996))を参照のこと。
前記Zuffereyらの論文は、HIV-1 env 遺伝子を含むpol の3’側配列が欠失しているレンチウイルスパッケージングプラスミドを示している。この構築物は、tat 及びrev 配列を含み、かつ3’側LTR はポリA配列で置換されている。5’側LTR 及びpsi 配列は、誘導性であるもののような別のプロモーターによって置換されている。例えば、CMV プロモーター又はそれらの誘導体を使用することができる。
【0036】
問題のパッケージングベクターは、レンチウイルスタンパク質の発現を増強しかつ安全性を増強するために、パッケージング機能を更に変更することを含む。例えばgag の上流のHIV 配列は全て除去することができる。更にenv の下流の配列も除去することができる。更に、RNA のスプライシング及び翻訳を増強するために、ベクターを修飾する工程を行うことができる。
【0037】
複製コンピテントなレンチウイルスの作出の可能性がよりごくわずかであるベクターを提供するために、本発明は、転写機構によりウイルス発現を促進する調節タンパク質であるtat 配列が機能的に欠失されているようなレンチウイルスパッケージングプラスミドを提供する。その結果、tat 遺伝子が、一部又は全て欠失されるか、もしくは様々な点突然変異又は他の突然変異がtat 配列に生じ、遺伝子を非機能性にする。技術者は、tat 遺伝子を非機能性にする公知の技術を実践することができる。
【0038】
ベクターの構築並びに細胞のトランスフェクション及び感染に使用される技術は、当該技術分野において広範に実践されている。実施者は、特定の条件及び手順を説明する標準の手段材料を熟知している。しかし、便宜上下記の段落においてガイドラインを示す。
本発明のベクターの構築には、当該技術分野において周知の標準的連結及び制限法を用いる(Maniatisら、Molecular Cloning : A Laboratory Manual 、コールドスプリングハーバー、NY、1982年を参照のこと)。単離されたプラスミド、DNA 配列又は合成されたオリゴヌクレオチドは、切断し、望ましい形状に適合させかつ再連結する。
【0039】
位置特異的DNA 切断は、当該技術分野において理解される条件下、及び特別な場合は市販の制限酵素の製造業者の指定する条件下で、適当な制限酵素(又は酵素類)で処理することによって行われ、これについては、例えばNew England Biolabs 社の製品カタログを参照のこと。一般に、プラスミド又はDNA 配列約1μg を、約20μl のバッファー液中で酵素1ユニットで切断する。典型的には、制限酵素の過剰量を用いて、DNA 基質の完全な消化を確実にする。およそ37℃で約1〜2時間のインキュベーション時間が実行可能であるが、その変形も許容することができる。各インキュベーション後、フェノール/クロロホルムを用いる抽出によりタンパク質を除去し、その後エーテルで抽出し、かつ核酸を水性画分からエタノール沈殿により回収する。所望であるならば、切断された断片のサイズ分離を、常法を用いポリアクリルアミドゲル又はアガロースゲル電気泳動により行うことができる。サイズ分離の一般的説明は、Method of Enzymology、65:499-560(1980)に見ることができる。
【0040】
制限切断された断片は、50mM Tris(pH7.6)、50mM NaCl 、6mM MgCl2 、6mM DTT 及び5〜10μM dNTPの中で、20℃で約15〜25分のインキュベーション時間を用いた、4種のデオキシヌクレオチド3リン酸(dNTP)の存在下における、E.coli DNAポリメラーゼIの巨大断片(クレノウ)による処理により平滑末端であり得る。クレノウ断片は、5’の粘着末端を満たすが、たとえ4種のdNTPが存在したとしても、突出している3’側の1本鎖を砕いてしまう(chew back) 。所望であるならば、選択性の修復が、粘着末端の性質によって指示された制約内で、唯一のdNTPの、又は選択されたdNTPの供給によって行うことができる。クレノウによる処理後、この混合物はフェノール/クロロホルムで抽出され、エタノール沈殿される。適当な条件下でのS1ヌクレアーゼ又はBal-31による処理は、いずれかの1本鎖部分の加水分解を生じる。
【0041】
連結は、下記の標準条件及び温度下で、容量15〜50μl の中で行うことができる:20mM Tris-Cl pH7.5、10mM MgCl2、10mM DTT、33mg/ml BSA 、10mM-50mM NaCl、及び40μM ATP 、0.01〜0.02(Weiss)ユニットのT4 DNAリガーゼを用い0℃(「粘着末端」の連結)又は1mM ATP 、0.3 〜0.6(Weiss)ユニットのT4 DNAリガーゼを用い14℃(「平滑末端」の連結)のいずれか。分子内「粘着末端」の連結は、通常総DNA 濃度33〜100 μg/mlで行われる(総末端濃度5〜100mM )。分子内平滑末端の連結は、(通常10〜30倍のモル過剰量のリンカーを用いて)1μM の総末端濃度で行われる。
【0042】
従って、本発明において、レンチウイルスパッケージングベクターは、プロモーター及び、gag 、pol 、rev 、env 又はそれらの組合せ、並びにtat の特異的機能の又は実際の削除を伴うような、当業者によって決定された他の任意又は必要な調節配列、及び任意に他のレンチウイルスアクセサリー遺伝子を含むように作出される。
【0043】
レンチウイルス導入ベクター(Naldini ら、前記;Proc. Natl. Acad. Sci.、93:11382-11388(1996))は、in vitroにおいてヒト細胞を増殖抵抗性(growth-arrested) に感染するために、及び成体ラットの脳へ直接注射した後に神経を形質導入するために使用されている。このベクターは、in vivo における神経へのマーカー遺伝子導入時の効率がよく、かつ検出可能な病理が存在しない場合に長期間発現が達成される。これまで試験した中で最長である該ベクターの単回注射後10ヶ月間にわたって分析した動物は、導入遺伝子発現の平均レベルにおいて減少を示さず、かつ組織病理又は免疫反応の徴候を示さなかった(Blomerら、J. Virol. 、71:6641-6649(1997))。非分裂細胞を形質導入するベクターの能力を損なうことなく、HIV ビルレンス遺伝子であるenv 、vif 、vpr 、vpu 及びnef が欠失されたレンチウイルスベクターの改善されたバージョンが開発されている。多様に減弱されたバージョンは、該ベクターの生体安全性の実質的改善を示している(Zuffereyら、前記)。
【0044】
形質導入された細胞においては、組込まれたレンチウイルスベクターは、一般に各末端にLTR を有する。5’側LTR は、特にHIV-感染細胞において、組換えの基質となり得る「ウイルス性」転写物の蓄積を引き起こすことがある。3’側LTR は、細胞のプロトオンコジーンを活性化した結果生じる危険性を伴う下流の転写を促進する。
【0045】
U3配列は、HIV LTR の大部分を含む。このU3領域は、感染細胞における及び細胞活性化に反応したHIV ゲノムの基本的かつ誘導された発現を変調するエンハンサーエレメント及びプロモーターエレメントを含む。プロモーターエレメントのいくつかが、ウイルス複製には必須である。エンハンサーエレメントの一部は、ウイルス単離体において高度に保存され、かつウイルス病原性の重大なビルレンス因子であることが示されている。エンハンサーエレメントは、ウイルスの様々な標的細胞において、複製率に影響を及ぼすように作用することができる(Marthas ら、J. Viol.、67:6047-6055(1993))。
【0046】
ウイルス転写は5’側LTR のU3領域の3’末端から始まるので、これらの配列は、ウイルスmRNAの一部ではなく、かつ3’側LTR 由来のそれらのコピーは、組込まれたプロウイルスにおいて両LTR 生成の鋳型として作用する。U3領域の3’コピーがレトロウイルスベクター構築物において変更される場合は、このベクターRNA は依然プロデューサー細胞の完全な5’側LTR から産生されるが、標的細胞において再生することはできない。このようなベクターの形質導入は、子孫ウイルスにおいて両LTR の不活性化を生じる。従ってレトロウイルスは、自己不活性(SIN) であり、かつこれらのベクターはSin 導入ベクターとして公知である。
【0047】
しかしながら、3’側LTR の欠失の程度には制限がある。第一に、U3領域の5’末端は、組込みに必要である、ベクター導入における他の本質的機能を提供する(末端のジヌクレオチド+att 配列)。従って末端ジヌクレオチド及びatt 配列は、欠失されているU3配列の5’側境界を示すことができる。加えて、一部の曖昧に定義された領域は、R領域下流のポリアデニル化部位の活性に影響を及ぼすことができる。3’側LTR のU3配列の過剰な欠失は、プロデューサー細胞におけるベクターの力価及び標的細胞における導入遺伝子の発現の両方に対し、有害な結果を伴うベクター転写物のポリアデニル化を減少することができる。他方、限定された欠失は、形質導入された細胞においてLTR の転写活性を無効にすることはできない。
【0048】
本願明細書において説明したレンチウイルス導入ベクターの新規バージョンでは、3’側LTR のU3領域の増加する欠失を保持している(図1:U3 LTRのヌクレオチド-418から指定された位置までのU3欠失の長さ:SIN-78、SIN-45、SIN-36及びSIN-18)。プロデューサー細胞におけるベクターの力価及び標的細胞における導入遺伝子の発現のいずれも損なうことなく、3’側LTR からのU3配列のほぼ完全な欠失を有するレンチウイルスベクターが開発された。最も広範な欠失(-418から-18 )は、TATAボックスにまで及び、その結果形質導入された細胞のLTR の転写活性が無効になっている。従って、3’側の欠失に関するより低い限界は、TATAボックスを含む位置まで及ぶ。この欠失は、R領域までのU3領域の残りである。これは、ベクターの安全性が劇的に増すことを意味している。様々な欠失が当該技術分野において公知の実施法により作出された。
【0049】
驚くべきことに、導入遺伝子の平均の発現レベルは、より完全なベクターと比べて、SIN ベクターによって形質導入された細胞においてでさえ高かった。これは恐らく、内部プロモーター上の上流HIV LTR から転写の妨害が除かれたためであろう。このようなU3領域の広範囲の欠失を伴うSIN-型ベクターは、形質導入効率を損なうことなく、マウス白血病ウイルス(MLV) を基にしたレトロウイルスベクターについて作出することはできない。
【0050】
導入ベクターの5’側LTR は、異種エンハンサー/プロモーターを伴うU3領域の転写調節エレメントの一部又は全部を置換することによって修飾された。この変更は、プロデューサー細胞における導入ベクターRNA の発現を増強するように;HIV のtat 遺伝子不存在下でのベクターの作製を可能にするように;並びに、前述のSIN ベクターを「救出」するための3’側が欠失したバージョンと再結合することができる上流のHIV LTR の野生型コピーを除去するように作出された。
【0051】
従って、5’側LTR に前述の変更を含むベクターである5’ベクターは、発現を増強する配列及びtat を発現しないパッケージング細胞との組合せのために、導入ベクターとしての使用を見出すことができる。
このような5’ベクターは、更に先に説明したように3’側LTR に修飾を保持することもでき、単に発現が増強されず、tat を発現しないパッケージング細胞において使用することができるだけでなく、更に自己不活性化することができるような改善された導入ベクターを生じる。
【0052】
HIV LTR からの転写は、tat タンパク質のトランスアクチベーター機能に高度に依存している。プロデューサー細胞に存在するコアパッケージング構築物によってしばしば発現されるtat が存在する場合、HIV LTR からのベクターの転写は強力に刺激される。完全な長さの「ウイルス」RNA は、パッケージングシグナルの完全な相補性を有しているので、このRNA は効率的にベクター粒子中でキャプシド形成され、かつ標的細胞に導入される。プロデューサー細胞のパッケージングによって利用できるベクターRNA の量は、感染性ベクター作製の律速段階である。
【0053】
5’側LTR のエンハンサー領域又はエンハンサー及びプロモーター領域は、各々、ヒトサイトメガロウイルス(CMV) 又はマウスラウス肉腫ウイルス(RSV) のエンハンサー又はエンハンサー及びプロモーターにより置換された。該構築物の概略及びハイブリッドベクターのコード名については、図2を参照のこと。CCL 及びRRL ベクターは、5’側U3領域が完全に置換されている。
【0054】
対照のレンチベクターHR2 及び5’側ハイブリッドのパネルを、導入ベクターによりトランスフェクションされたプロデューサー細胞において、tat トランスアクチベーターを提供するパッケージング構築物の存在下又は不存在下で、比較した。4種のキメラベクターの転写レベルは、パッケージング構築物の存在下及び不存在下の両方で、対照レンチベクターのそれよりも高い。全てのキメラベクターは、導入遺伝子を標的細胞に効率的に導入し、かつRRL ベクターは対照HR2 ベクターと同様に作用する。最後に、ベクターの標的細胞への組込みを、形質導入後の初期及び後期継代時に形質導入された細胞を調べることにより確認した。そのベクターが組込まれたことを示す導入遺伝子−陽性細胞の割合に減少は認められなかった。
【0055】
パッケージング構築物不存在下でプロデューサー細胞において得られた5’側LTR が修飾された導入ベクターRNA の高レベルの発現は、作製されるベクターが機能性tat 遺伝子の不存在下で機能していることを示している。先に本願明細書において説明したパッケージングプラスミドについて示されたtat 遺伝子の機能欠損は、tat タンパク質に関連した多くの病理活性が授けられたレンンチウイルスベクターシステムに対するより高レベルの生体安全性をもたらすであろう。従って、生体安全性が顕著に改善されたレンチウイルスベクターは、5’末端又は3’末端のいずれにもHIV LTR の野生型コピーを有さないSIN 導入ベクターであり、これは本願明細書において説明したtat −不含有パッケージングベクターと共同して使用される。
【0056】
ウイルス上清は、トランスフェクションの48時間後の上清のろ過のような常法を用いて回収される。ウイルスの力価は、例えば適量のウイルスの上清による、106 個のNIH 3T3 細胞又は105 個のHeLa細胞の感染により、ポリブレン(シグマケミカル社、セントルイス、MO)8μg/mlの存在下で測定される。48時間後、形質導入効率がアッセイされる。
【0057】
このように本発明は、高力価組換えウイルスを作製する方法及び手段を提供する。このようなウイルス粒子調製物を用いて、当該技術分野において公知の方法により標的細胞を感染することができる。従って本発明は、標的細胞が宿主から取り出され、公知の方法を行い培地において形質転換され、その後該宿主に戻されるような、ex vivo 遺伝子治療の用途における使用を見出すであろう。
【0058】
以下に記された本発明の詳細な説明は、本発明の様々な実施態様を示す限定を意図しない実施例である。
実施例1
レンチウイルスパッケージングプラスミドの構築
レンチウイルスパッケージングプラスミドは、前記Zuffereyらの論文において先に記されたプラスミドpCMVΔR8.9(ΔVpr ΔVif ΔVpu ΔNef )に由来した。pCMVΔR8.9のnef 遺伝子の残りの配列を、XhoI及びBstEIIによる消化により除去し、クレノウで満たし、かつ再連結した。この構築物の、100 塩基対を欠失し、HIV-1 の切断型env のリーディングフレームを、ゲノムインスリンのポリアデニル化部位に結合し、プラスミドpCMVΔR8.73 を得た。
【0059】
本発明の別の実施態様において、プラスミドpCMVΔR8.73 は、CMV プロモーターの下流のCMV 由来の配列の133 塩基対が欠失されている。この配列は、スプライスドナー部位を含み、かつこれはプラスミドpCMVΔR8.73 のSacII による消化及びより大きい断片への再連結によって除去され、プラスミドpCMVΔR8.74 が得られた。
【0060】
別の本発明の実施態様においては、gag 遺伝子の開始コドンの上流のプラスミドpCMVΔR8.74 に残っているHIV-由来配列の、コンセンサス5’スプライスドナー部位以外の全てを除去した。同時に、gag 遺伝子の上流の配列を、最適翻訳効率で変更し、プラスミドpCMVΔR8.75 を得た。pCMVΔR8.75 は、pCMVΔR8.74 に由来し、94塩基対のSstII-ClaI断片を、以下から成るSstII-ClaIオリゴヌクレオチドリンカーで置き換えることによって得た:5’-GGGACTGGTGAGTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3’(配列番号:1)及び5’-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACCCATGGCGGCGGCAGATCTCGAATTCACTCACCAGTCCCGC-3’(配列番号:2)。
【0061】
別の本発明の実施態様において、誘導可能なパッケージング構築物が、CMV プロモーターを含むpCMVΔR8.74 のPstI-SacII断片を、最小のCMV プロモーターに連結したテトラサイクリンオペレーター配列の7個のタンデムコピーと置き換えることにより得た。tet-調節されたパッケージングプラスミドpTetΔR8.74 を得た。
実施例2
レンチウイルス導入ベクターの構築
レンチウイルス導入ベクタープラスミドは、先にNaldini らの論文(Sci.、272:263-267(1996) )に記されたプラスミドpHR'-CMV-LacZ に由来した。pHR2は、pHR'中の3’側LTR の上流のnef 配列の 124塩基対が、HIV1配列を減少しかつ導入遺伝子のクローニングが促進されるように両方のポリリンカーと置き換えられているような、レンチウイルス導入ベクターである。pHR2は、pHR'-CMV-LacZ に由来し、4.6kb ClaI-StuI 断片を、鋳型としてpHR'-CMV-LacZ を用いかつオリゴヌクレオチドとして下記を使用するPCR により作製された 828塩基対のClaI-StuI 断片の、pHR'-CMV-LacZ 由来の4.4kb StuI-NcoI 断片及び4.5kb NcoI-ClaI 断片と3部位で連結したものと置き換えることによって得た:5’-CCATCGATCACGAGACTAGTCCTACGTATCCCCGGGGACGGGATCCGCGGAATTCCGTTTAAGAC-3’(配列番号:3)及び5’-TTATAATGTCAAGGCCTCTC-3’(配列番号:4)。
【0062】
本発明の別の実施態様において、pHR3は、pHR2のRev 反応エレメント(RRE) の上流のenv コード配列の 148塩基対(ATG を含む)が欠失しているようなレンチウイルスベクターである。pHR3はpHR2に由来し、pHR2の 839塩基対NotI-SpeI 断片を、鋳型としてpHR2を用いかつオリゴヌクレオチドプライマーとして下記を使用するPCR により得られた 747塩基対NotI-SpeI 断片と置き換えることによって得た:5’-GCGGCCGCAGGAGCTTTGTTCCTTGG-3’(配列番号:5)及び5’-TACGTAGGACTAGTCTCG-3’(配列番号:6)。
【0063】
本発明の別の実施態様において、pRH5は、 310塩基対gag コード配列(Gag タンパク質の15番目のアミノ酸の下流の全gag コード配列)がpHR2から欠失されているレンチウイルス導入ベクターである。pHR5は、pHR2のNruIによる消化、NotIリンカー(合成オリゴヌクレオチド5’-TTGCGGCCGCAA-3’、(配列番号:7))の付加、 310塩基対の断片を切り出すためのNotIによる消化、その後の再連結によって得た。
【0064】
本発明の別の実施態様において、pRH6は、パッケージすることが可能な完全な長さの転写物の産生を増強するために、5’側スプライスドナーシグナルが変異された(TGGTがTGATへ)レンチウイルスベクターである。pHR6はpHR5に由来し、 239塩基対AflII-ApoI断片を、鋳型としてpHR2を用いかつオリゴヌクレオチドプライマーとして下記を使用するPCR により作製された 239塩基対AflI-ApoI 断片によって置き換えることによって得た:5’-CCACTGCTTAAGCCT-3’(配列番号:8)及び5’-CAAAATTTTTGGCGTACTCATCAGTCGCCGCCCCTCG-3’(配列番号:9)。
【0065】
全てのPCR 断片は、直接TAクローニングベクターpCR2.1(インビトロゲン社)へのPCR 反応産物の最初のクローニング、それに続く配列の検証及び適当な酵素による切り出しにより作製した。
実施例3
5’側LTR キメラレンチウイルス導入ベクターの構築
本発明の別の実施態様において、レンチウイルスベクターの5’側LTR は、HIV-1 のR領域に結合したラウス肉腫ウイルス(RSV) のU3領域由来のエンハンサー及びプロモーターを含む(プラスミドpRRL)。pRRLは、オリゴヌクレオチドリンカーを用いて、RSV のエンハンサー及びプロモーター(転写開始位置に対してヌクレオチド-233から-1)がHIV-1 のR領域に正確に融合されたようなレンチウイルス導入ベクターである。pRRLは、国際公開公報第97/07225号を参照し、プラスミドpRT43.RSV.F3及びpRH2に由来し、かつpRT43.RSV.F3の3.4kb EcoRI-HpaI断片を、pHR2の0.67kb BglII-NotI 断片及びpHR2の1.7kb NotI-StuI 断片で、5’-AATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGGGTCTCTCTGGTTAGACCA-3’(配列番号:10)及び5’-GATCTGGTCTAACCAGAGAGACCCGTTTATTGTATCGAGCTAGGCACTTAAATACAATATCTCTGCAATGCGGC-3’(配列番号:11)のオリゴヌクレオチドからなる合成EcoRI-BglII オリゴヌクレオチドリンカーにより置き換えることによって得た。
【0066】
本発明の別の実施態様において、レンチウイルスベクターの5’側LTR は、HIV-1 のプロモーター領域(転写開始位置に対して -78塩基対の位置から)に結合したラウス肉腫ウイルス(RSV) のエンハンサー(転写開始位置に対してヌクレオチド-233から-50 )を含む(プラスミドpRLL)。
pRLLは、RSV のエンハンサーがHIV-1 のプロモーター領域にオリゴヌクレオチドリンカーを用いて融合されたレンチウイルス導入ベクターである。pRRLは、プラスミドpRT43.RSV.F3及びpHR2に由来し、かつpRT43.RSV.F3の3.4kb EcoRI-HpaI断片を、pRH2の0.724kb AlwNI-NotI断片及びpRH2の1.7kb NotI-StuI 断片で、オリゴ5’-AATTGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3’(配列番号:12)及びオリゴヌクレオチド5’-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCC-3’(配列番号:13)からなる合成EcoRI-AlwNI オリゴヌクレオチドリンカーにより置き換えることによって得た。
【0067】
本発明の別の実施態様(プラスミドpCCL)において、レンチウイルスベクターの5’側LTR は、HIV-1 のR領域に結合した、ヒトサイトメガロウイルス(CMV) の直前の初期(immediate early)エンハンサー及びプロモーター(Boshart らの発表した(Cell、41:521-530(1985))転写開始位置に対してヌクレオチド-673から-1)を含む。pCCLは、プラスミドpRT43.2F3 (米国特許第5,686,279 号)及びpHR2に由来し、かつpRT43.2F3 の3.8kb SstI-HpaI 断片を、pRH2の1.7kb BglII-NotI断片及びpRH2の1.7kb NotI-StuI 断片で、オリゴヌクレオチド5’-CGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA-3’(配列番号:14)及び5’-GATCTGGTCTAACCAGAGAGACCCCGGTTCACTAAACGAGCT-3’(配列番号:15)からなる合成SstI-BglIIオリゴヌクレオチドリンカーにより置き換えることによって得た。
【0068】
本発明の別の実施態様(プラスミドpCLL)において、レンチウイルスベクターの5’側LTR は、HIV-1 のプロモーター領域(転写開始位置に対して -78塩基対位から)に結合したサイトメガロウイルス(CMV) の転写開始位置に対して-220から-673のエンハンサーヌクレオチドを含む。pCLLは、プラスミドpRT43.2F3 及びpHR2に由来し、かつpRT43.2F3 の3.6kb NcoI-HpaI 断片を、pRH2の0.724kb AlwNI-NotI断片及びpRH2の1.7kb NotI-StuI 断片で、オリゴ5’-CATGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3’(配列番号:16)及び5’-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTC-3’(配列番号:17)からなる合成NcoI-AlwNIオリゴヌクレオチドリンカーにより置き換えることによって得た。
実施例4
自己不活性化するレンチウイルスベクターの構築
pRRL.SIN-18 はpRRLに由来し、消化及び再連結により3’ LRTにおける 400塩基対EcoRV-PvuII 断片が欠失された。
【0069】
pRRL.SIN-36 はpRRLに由来し、3’側LTR の 493塩基対BbsI-AlwNI断片を、合成オリゴヌクレオチド5’-GATATGATCAGATC-3’(配列番号:18)及び5’-CTGATCA-3’並びにpRRL由来の0.54kb AlwN-AvrII 断片及び6.1kb AverII-BbsI 断片との3部位の連結からなるオリゴヌクレオチドリンカーにより置き換えることによって得た。
【0070】
pRRL.SIN-45 はpRRLに由来し、3’側LTR の 493塩基対BbsI-AlwNI断片を、合成オリゴヌクレオチド5’-GATATGATCAGAGCCCTCAGATC-3’(配列番号:19)及び5’-CTGAGGGCTCTGATCA-3’(配列番号:20)並びにpRRL由来の6.1kb AlwNI-AvrII 断片及び6.1kb AverII-BbsI 断片との3部位の連結からなるオリゴヌクレオチドリンカーにより置き換えることによって得た。
【0071】
pRRL.SIN-78 はpRRLに由来し、3’側LTR の 493塩基対BbsI-AlwNI断片を、5’-GATATGATCAGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3’(配列番号:21)及びオリゴヌクレオチド5’-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCCTGATCA-3’(配列番号:22)並びにpRRL由来の0.54kb AlwNI-AvrII断片及び6.1kb AverII-BbsI 断片との3部位の連結からなるオリゴヌクレオチドリンカーにより置き換えることによって得た。
実施例5
安定したレンチウイルスパッケージング細胞00-28 の構築及びレンチウイルスベクターの安定した産物
293G細胞株を用いて、安定したレンチウイルスパッケージング細胞を作製した。293G細胞は、MDカセット(CMV プロモーター、並びにヒトβグロビン遺伝子由来の介入配列−エクソン2及び3、イントロン2-及びポリ(A) 部位)からのtet R /VP16 トランスアクチベーター、並びに7個のテトラサイクリンオペレーター部位(tetO )の縦列反復配列に連結した最小のCMV プロモーター由来のVSV エンベローブを発現する。従って、VSV G の発現は、培養培地のテトラサイクリン濃度により調節され、この抗生物質が存在する場合には抑制される(Gossen及びBujard、Proc. Natl. Acad. Sci. USA、89:5547-5551(1992);Ory ら、Proc. Natl. Acad. Sci. USA、93:11400-11406(1997))。293G細胞は、通常10%ドナーウシ血清を補充し、かつ1μg/mlテトラサイクリンを含有するDMEM/低濃度グルコース培養培地中で維持される。293G細胞の15cmプレートを、リポフェクタミン(GIBCO BRL) を用い、パッケージングプラスミドpCMVΔR8.74 の13.36 μg 及び選択プラスミドpZeoSV2 の1.33μg で、トランスフェクションした。この培地を24時間後に交換し、48時間後に細胞を、ゼオシン250 μg/ml及びテトラサイクリン1μg/mlを含有する培地に分けた。3〜4週間選択した後、250 クローンを採取し、96ウェルプレートに移し、かつこの培地を市販のキットを用いて、免疫捕獲によりHIV-1 p24 Gag 抗原についてスクリーニングした。52個のp24 陽性クローンを更なる分析のために増殖した。最良の5個のクローンが、12〜23ng/ml のp24 値を有することが決定された。これらの5個のクローンのうち4個は、ウェスタンブロット分析により、テトラサイクリン除去後、VSV.G 発現について陽性であった。
【0072】
更に4個のp24/VSV.G 陽性クローンを、レンチウイルス導入ベクターをパッケージする能力について分析した。これらのクローンを、CMV プロモーターで起動されたA. Victoria のグリーン蛍光タンパク質(GFP) の発現カセットを含む一過性に産生されたレンチウイルスベクター(VSV.G 偽型)で、感染多重度10かつポリブレン(8μg/ml)存在下で、感染した。次に感染したクローンを拡張し(expanded)、テトラサイクリンを除去した。誘導の72時間後、24時間培地の収集を行い、上清をろ過し、急速凍結した。凍結した上清を、GFP 遺伝子の形質導入について天然のHeLa細胞上で力価測定した。FACS分析により、パッケージングクローン00-28 の感染によって生じた細胞集団(10-28 と称される)は、最大の力価5×104 形質導入単位(T.U.)/ml を有することが決定された。
【0073】
感染されたパッケージング集団10-28 を、GFP レンチウイルスベクターの高力価産生クローンの作出に用いた。10-28 細胞は、FACSにより調べ、かつ最高のGFP 発現細胞を保持し拡張した。その後この集団を、一過性に作製されたGFP レンチウイルス(VSV.G 偽型)で更に4回、連続的に(「ピング(ping)」)感染した。各々感染した後、上清をVSV.G 誘導の72〜96時間後に収集した。上清を、HeLa細胞上で力価決定し、かつ免疫捕獲アッセイによりp24 含量について分析した。感染力価は、3回目のピング後にピークを示し、1.5 ×106T.U./mlに達した(図3参照)。3回目のピングからの細胞集団を、サブクローニングし、高力価ベクターのプロデューサーを単離した。
【0074】
本願明細書に引用した論文及び特許出願は全て、個々の論文又は特許出願が明確かつ個別に参照として組込まれることが示されるように、それらの全体が本願明細書に参照として組込まれている。
本発明に関連する当業者には明らかであるように、本発明は、先に明確に示されたもの以外の形、例えば、他の哺乳類細胞型のトランスフェクション及び形質導入も、本発明の精神又は本質的特長から逸脱することがない限りは包含している。従って前述の本発明の特定の実施態様は、例証のためであり制限するものではないとみなすべきである。本発明の範囲は、記したように、前述の説明において限定された実施例に制限されるものではなく、添付された特許請求の範囲である。
【図面の簡単な説明】
【図1】 これは様々なレンチウイルスベクターを示す。RSV はラウス肉腫ウイルスエンハンサー/プロモーターであり;RはLTR のR領域であり;U5はLTR のU5領域であり;SDはスプライスドナー部位であり、例えばHIV 5’主要スプライスドナー部位であり:ψはプサイキャプシド形成シグナル配列であり;Gaはgag 遺伝子の一部であり;RRE はrev 反応性エレメントであり;SAはスプライスアクセプター配列であり;U3はLTR のU3領域である。
【図2】 これは、別のレンチウイルスベクターを示す。CMV はサイトメガロウイルスである。他の点では、記号は図1に示したものと同じである。
【図3】 これは、導入ベクターの量を漸増する場合の等級化されたベクター産生を示すグラフである。
【配列表】
[0001]
Field of Invention
The present invention relates to novel lentiviral packaging vectors, transduction vectors carrying foreign genes of interest, stable packaging cell lines, stable producer cell lines, and their production for producing recombinant lentiviruses in mammalian cells. Regarding use.
Background of the Invention
Retroviral vectors are a common tool for gene delivery (Miller, Nature, 357: 455-460 (1992)). The ability of retroviral vectors to deliver unrearranged single copy genes to a wide range of rodent, primate and human somatic cells creates retroviral vectors well suited for gene transfer into cells.
[0002]
A useful accessory for creating recombinant retroviral vectors is a packaging cell line that supplies the proteins required for the production of infectious virions in trans, but such cells are endogenous. Cannot be packaged (Watanabe and Temin, Molec. Cell Biol., 3 (12): 2241-2249 (1983); Mann et al., Cell, 33: 153-159 (1983) Embretson and Temin, J. Viol., 61 (9): 2675-2683 (1987)). Considerations for the construction of retroviral packaging cell lines include rodents (Cloyd et al., J. Exp. Med., 151: 542-552 (1980)) and primates (Donahue et al., J. Exp. Med., 176 : 1125-1135 (1992)), production of high titer vector supernatants free of recombinant replication competent retrovirus (RCR), which has been shown to produce T lymphocytes.
[0003]
One way to minimize the possibility of RCR generation in packaging cells is to use a packaging function that expresses two genomes, eg, one that expresses the gag and pol gene products and the other that expresses the env gene product. (Bosselman et al., Molec. Cell Biol., 7 (5): 1797-1806 (1987); Markowitz et al., J. Virol., 62 (4): 1120-1124 (1988); Danos and Mulligan, Proc. Natl. Acad. Sci., 85: 6460-6464 (1988)). This method highlights the recombination frequency due to the ability of simultaneous packaging and subsequent transfer of these two genomes, as well as the presence of three retroviral genomes in the packaging cell to produce RCR. Minimize the ability to decrease.
[0004]
In the event that a recombinant occurs, mutations (Danos and Mulligan, supra) or deletions (Boselman et al., Supra) or Markowitz et al. Can be designed. In addition, deletion of the 3 'LTR on both packaging constructs also reduces the ability to form functional recombinants.
[0005]
Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the general retroviral genes gag, pol and env. The high degree of complexity allows the lentivirus to modulate its life cycle during the latent infection period.
A typical lentivirus is the human immunodeficiency virus (HIV), a pathogen of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages. In vitro, HIV can infect HeLa-Cd4 cells or T lymphocyte cells whose cell cycle has been suppressed by primary culture of monocyte-derived macrophages (MDM) and further by treatment with aphidicolin or γ-irradiation. it can.
[0006]
Cell infection depends on active transport of the pre-integration complex of HIV into the nucleus through the nuclear pore of the target cell. This is caused by the mechanism of transport of the target cell into the nucleus and the interaction of multiple partially overlapping molecular determinants in the complex. The determinants identified are the functional nuclear localization signal (NLS) of the C-terminal phosphotyrosine residue of the gag matrix (MA) protein, the karyophilic virion-related protein, vpr, and the gag MA protein including.
Summary of the Invention
Thus, the present invention provides a novel safe lentiviral vector related to the synthesis of both lentiviral vector transcripts that can be packaged and lentiviral proteins for the rapid production of high titer recombinant lentiviruses in mammalian cells. About. These results are infectious particles for delivering the foreign gene in question to target cells. The invention further provides cell lines for virus production.
Detailed Description of the Invention
The present invention provides a recombinant lentivirus capable of infecting non-dividing cells, as well as methods and means for producing the same. This virus is useful for the introduction and expression of nucleic acid sequences in vivo and ex vivo.
[0007]
The lentiviral genome and proviral DNA have three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeats (LTR). The gag gene encodes internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), protease and integrase; and the env gene Encodes the envelope glycoprotein. The 5 'and 3' LTRs can be used to promote virion RNA transcription and polyadenylation. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx (HIV-1, HIV-2 and / or SIV).
[0008]
Adjacent to the 5 'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and sequences necessary for effective encapsidation of viral RNA particles (psi site). This cis deficiency prevents encapsidation of genomic RNA if the sequences necessary for encapsidation (or packaging of retroviral RNA into infected virions) are lost from the viral genome. However, the resulting mutants can still direct the synthesis of all virion proteins.
[0009]
The present invention is capable of infecting non-dividing cells, including transfection into suitable host cells with two or more vectors having packaging functions, i.e. gag, pol and env, as well as rev and tat. A method for producing a recombinant lentivirus is provided. As described below, vectors lacking a functional tat gene are desirable for specific applications. Thus, to produce a packaging cell, for example, the first vector can provide a nucleic acid encoding viral gag and viral pol, and another vector encodes viral env. Nucleic acid can be provided. Incorporation of a vector providing a heterologous gene identified herein as a transfer vector into a packaging cell results in a producer cell that releases infectious viral particles that carry the foreign gene of interest.
[0010]
The aforementioned vectors themselves are outside the scope of the newly constructed vectors disclosed herein, but this is known in the art and is described in Naldini et al. (Sci., 272: 263-267). (1996)); and Zufferey et al. (Nat. Biotech., 15: 871-875 (1997)). In general, these vectors are plasmid-based or virus-based and are designed to retain sequences essential for the incorporation of foreign nucleic acids, selection of nucleic acids, and introduction into host cells. The gag, pol and env genes of the vector in question are also known in the art. The relevant gene is therefore cloned into the selected vector and then used for transformation of the target cell in question.
[0011]
In accordance with the vector and foreign gene design described above, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from any virus, including retroviruses. Preferably env is an amphotropic envelope protein that allows transduction of cells of human and other species.
[0012]
It is desirable to target the recombinant virus with a specific ligand for binding of this envelope protein to an antibody or targeting to a receptor of a specific cell type. By inserting the sequence of interest (including regulatory regions) into the viral vector, eg, with other genes encoding receptor ligands on specific target cells, the vector becomes target specific. Retroviral vectors can be made target specific, for example, by insertion of glycolipids or proteins. Targeting is accomplished by using an antigen-binding moiety such as an antibody or a recombinant antibody-type molecule, such as a single chain antibody, and often targets retroviral vectors. Those skilled in the art will know specific methods for achieving delivery of retroviral vectors to specific targets or will readily ascertain without undue experimentation.
[0013]
Examples of retrovirus-derived env genes include Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), mouse breast cancer virus (MuMTV or MMTV), Gibbon monkey leukemia virus (GaLV or GALV), human Including but not limited to immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes such as vesicular stomatitis virus (VSV) protein G (VSVG), hepatitis virus and influenza can also be used.
[0014]
Vectors that provide viral env nucleic acid sequences are operably linked to regulatory sequences such as promoters or enhancers. The regulatory sequence can be a eukaryotic promoter or enhancer, including, for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer element is located within or adjacent to the LTR sequence.
[0015]
Preferably, the regulatory sequence is not endogenous to the lentivirus from which the vector was constructed. Thus, if the vector is generated from SIV, the SIV regulatory sequences found in the SIV LTR will be replaced by regulatory elements that do not originate from SIV.
VSV G is a desirable env gene because VSV G imparts a broad host cell range to recombinant viruses, but VSV G can be detrimental to host cells. Therefore, if a gene such as the gene for VSV G is used, an inducible promoter system is used, so that if VSV G expression is not required, VSV G expression is regulated to minimize host toxicity. It is preferable to be able to do this.
[0016]
For example, tetracycline was removed from the introduced cells using the tetracycline-regulated gene expression system described in Gossen and Bujard (Proc. Natl. Acad. Sci., 89: 5547-5551 (1992)). In some cases it can be used to provide inducible expression of VSV G. Thus, the tet / VP16 transactivator is present on the first vector and the sequence encoding VSV G is cloned downstream of the promoter controlled by the tet operator sequence on another vector.
[0017]
A transgene that is a heterologous or foreign nucleic acid sequence is operably linked to a regulatory nucleic acid sequence. As used herein, the term “heterologous” nucleic acid sequence means that the sequence originates from a foreign species or, if derived from the same species, is substantially modified from its original form. Alternatively, an unchanged nucleic acid sequence that is not normally expressed in a cell is a heterologous nucleic acid sequence.
[0018]
The term “operably linked” means that the regulatory sequence and the heterologous nucleic acid sequence are operably linked, resulting in the latter expression. Preferably, the heterologous sequence is linked to a promoter, resulting in a chimeric gene. This heterologous nucleic acid sequence is preferably under the control of either a viral LTR promoter-enhancer signal or an internal promoter, and the signal retained in the retroviral LTR can still result in effective expression of the transgene. .
[0019]
The foreign gene can be any of the nucleic acids in question that can be transcribed. In general, a foreign gene encodes a polypeptide. This polypeptide preferably has some therapeutic benefit. The polypeptide can compensate for the deficiency or absence of endogenous protein expression in the host cell. This polypeptide can confer new properties to the host cell, such as the chimeric signal receptor disclosed in US Pat. No. 5,359,046. Engineers can determine the suitability of foreign genes by practicing the techniques described herein and known in the art. For example, the engineer will know whether the foreign gene is of a size suitable for encapsidation and whether the foreign gene product is properly expressed.
[0020]
It is desirable to modulate the expression of genes that regulate molecules in cells by the incorporation of molecules by the methods of the present invention. The term “modulation” describes the suppression of gene expression when overexpressed or increased expression when underexpressed. Where cell proliferation disorders are associated with gene expression, nucleic acid sequences that interfere with gene expression at the translational level can be used. This method involves the transcription of specific mRNA, for example by using an antisense nucleic acid ribozyme or triplex agent to hide the mRNA with a nucleic acid or triplex material or cleaving it with a ribozyme. Alternatively, translation can be inhibited.
[0021]
An antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule (Weintraub, Sci. Am., 262: 40 (1990)). In the cell, the antisense nucleic acid hybridizes to the corresponding mRNA and forms a double-stranded molecule. Since cells do not translate double-stranded mRNA, antisense nucleic acids interfere with mRNA translation. Antisense oligomers of about 15 nucleotides or more are preferred because they are easily synthesized and are likely to cause less problems than larger molecules when incorporated into target cells. The use of antisense methods to inhibit in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172: 289 (1988)).
[0022]
Antisense nucleic acids can be used to block the expression of mutant proteins such as amyloid precursor protein or dominant active gene products that accumulate in Alzheimer's disease. Such methods are also useful for the treatment of Huntington's disease, hereditary Parkinson's disease and other diseases. Antisense nucleic acids are also useful for inhibiting the expression of proteins associated with toxicity.
[0023]
The use of oligonucleotides to stop transcription can be by a mechanism known as a triplex strategy in which oligomers wrap around a double helix DNA and form a triple stranded helix. Thus, triplex compounds can be designed to recognize unique sites on selected genes (Maher et al., Antisense REs and Dev., 1 (3) 227 (1991); Helene, Anticancer Drug Dis ., 6 (6): 569 (1991)).
[0024]
Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNAs in a manner similar to DNA restriction endonucleases. Through modification of the nucleotide sequences encoding these RNAs, it is possible to manipulate molecules that recognize and cleave specific nucleotide sequences in RNA molecules (Cech, J. Amer. Med. Assn., 260: 3030). (1988)). The great advantage of such a method is that only mRNA with a specific sequence is inactivated.
[0025]
It is desirable to introduce a nucleic acid encoding a biological response modifier. Included in this category are immunostimulants that include nucleic acids encoding many cytokines that are classified as “interleukins”, such as interleukins 1-12. Also included in this category are interferons, particularly γ-interferon (γ-IFN), which do not necessarily act according to the same mechanism, but are tumor necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM- CSF). Desirably, such nucleic acids are delivered to bone marrow cells or macrophages to treat congenital enzyme deficiencies or immunodeficiencies. Nucleic acids encoding growth factors, toxic peptides, ligands, receptors or other physiologically important proteins can also be incorporated into specific non-dividing cells.
[0026]
Accordingly, the recombinant lentivirus of the present invention can be used to treat HIV-infected cells (eg, T cells or macrophages) with anti-HIV-molecules. In addition, for example, airway epithelium can be infected with a recombinant lentivirus of the invention having a cystic fibrosis transmembrane conductance regulatory (CFTR) gene for the treatment of cystic fibrosis.
[0027]
The methods of the present invention further include ex vivo transplantation of cells infected with the recombinant lentivirus of the present invention, or into the central nervous system or ventricular cavity or subdurally on the surface of the host brain. It is useful for transplantation or “grafting” of nerve cells, glial cells, fibroblasts or mesenchymal cells associated with infection in vivo. Methods for such grafting are well known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS (1985) edited by Bjorklund and Stenevi.
[0028]
For diseases caused by protein product deficiencies, gene transfer can incorporate normal genes into infected tissues for compensation therapy, and even create animal models for diseases using antisense mutations. . For example, for infection of muscle, spleen or liver cells, it is desirable to insert a nucleic acid encoding factor VIII or IX into the lentivirus.
[0029]
The promoter sequence can be homologous or heterologous to the desired gene sequence. A wide range of promoters are available, including viral or mammalian promoters. Cell or tissue specific promoters can be used to target expression of gene sequences in specific cell populations. Mammalian or viral promoters suitable for the present invention are available in the art.
[0030]
Optionally, during the cloning step, a nucleic acid construct referred to as a transfer vector having a packaging signal and a heterologous cloning site further comprises a selectable marker gene. The marker gene is used to check the presence of the vector, so that infection or integration is confirmed. The presence of the marker gene ensures selection and growth of only host cells expressing the insert. Typical selection genes encode proteins that confer resistance to antibiotics or other toxicants such as histidinol, puromycin, hygromycin, neomycin, methotrexate, and cell surface markers.
[0031]
The recombinant virus of the present invention can introduce a nucleic acid sequence into mammalian cells. The term “nucleic acid sequence” means any nucleic acid molecule, particularly DNA, as discussed in detail herein. Nucleic acid molecules can be derived from a variety of sources including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid sequences can include genomic DNA that may or may not include natural introns. Furthermore, such genomic DNA can be obtained in relation to the promoter region, poly A sequence or other related sequences. Genomic DNA can be extracted and purified from appropriate cells by means well known in the art. Alternatively, messenger RNA (mRNA) can be isolated from cells and used to make cDNA by reverse transcription or other means.
[0032]
Preferably, the recombinant lentivirus produced by the method of the present invention is a derivative of human immunodeficiency virus (HIV). This env will be derived from a virus other than HIV.
The methods of the present invention, in some embodiments, comprise three vectors that provide all the functions necessary for packaging of recombinant virions, such as gag, pol, env, tat and rev, as described above. provide. As noted herein, tat can functionally lack unexpected benefits. As long as these vectors are used for transformation and production of packaging cell lines and produce recombinant lentiviruses, there is no restriction on the number of vectors used.
[0033]
Such vectors are incorporated into packaging cell lines by transfection or infection. This packaging cell line produces viral particles containing the vector genome. Methods of transfection or infection are well known to those skilled in the art. After co-transfection of the packaging vector and the transfer vector into the packaging cell line, the recombinant virus is recovered from the culture medium and titered by conventional methods used by those skilled in the art.
[0034]
Therefore, this packaging construct is introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, usually with a dominant selectable marker such as neo, DHFR, Gln synthetase or ADA, and then Selection and clones can be isolated in the presence of an appropriate drug. These selectable marker genes can be physically linked to the packaging gene in the construct.
[0035]
Stable cell lines designed so that the packaging function is expressed by suitable packaging cells are known. See, for example, US Pat. No. 5,686,279 disclosing packaging cells; and Oray et al. (Proc. Natl. Acad. Sci., 93: 11400-11406 (1996)).
The Zufferey et al. Paper shows a lentiviral packaging plasmid lacking the 3 'sequence of pol containing the HIV-1 env gene. This construct contains the tat and rev sequences and the 3 'LTR is replaced with a poly A sequence. The 5 'LTR and psi sequences are replaced by alternative promoters such as those that are inducible. For example, a CMV promoter or a derivative thereof can be used.
[0036]
The packaging vector in question involves further modifying the packaging function to enhance lentiviral protein expression and enhance safety. For example, all HIV sequences upstream of gag can be removed. In addition, sequences downstream of env can be removed. Furthermore, a step of modifying the vector can be performed to enhance RNA splicing and translation.
[0037]
In order to provide a vector with less potential for the production of replication-competent lentiviruses, the present invention provides a functional deletion of the tat sequence, a regulatory protein that promotes viral expression by a transcriptional mechanism. Such lentiviral packaging plasmids are provided. As a result, the tat gene is partially or completely deleted, or various point mutations or other mutations occur in the tat sequence, rendering the gene non-functional. Engineers can practice known techniques for rendering the tat gene non-functional.
[0038]
The techniques used for vector construction and cell transfection and infection are widely practiced in the art. The practitioner is familiar with standard instrumental materials that describe specific conditions and procedures. However, guidelines are provided in the following paragraph for convenience.
Standard ligation and restriction methods well known in the art are used to construct the vectors of the invention (see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982). The isolated plasmid, DNA sequence or synthesized oligonucleotide is cleaved, adapted to the desired shape and religated.
[0039]
Site-specific DNA cleavage is performed by treatment with an appropriate restriction enzyme (or enzymes) under conditions understood in the art and, in particular, as specified by the manufacturer of a commercially available restriction enzyme. For example, see the New England Biolabs product catalog. In general, about 1 μg of plasmid or DNA sequence is cleaved with 1 unit of enzyme in about 20 μl of buffer solution. Typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of approximately 1-2 hours at approximately 37 ° C. are feasible, but variations thereof are acceptable. After each incubation, the protein is removed by extraction with phenol / chloroform, followed by extraction with ether, and the nucleic acid is recovered from the aqueous fraction by ethanol precipitation. If desired, size separation of the cleaved fragments can be performed by polyacrylamide gel or agarose gel electrophoresis using conventional methods. A general description of size separation can be found in Method of Enzymology, 65: 499-560 (1980).
[0040]
Restricted fragments were 50 mM Tris (pH 7.6), 50 mM NaCl, 6 mM MgCl.2 Of E. coli DNA polymerase I in the presence of 4 deoxynucleotide triphosphates (dNTPs) in 5 mM, 10 mM DTT and 5-10 μM dNTPs using an incubation time of about 15-25 minutes at 20 ° C. It can be blunt ended by treatment with a large fragment (Klenow). The Klenow fragment fills the 5 'sticky end, but breaks the protruding 3' single strand even if four dNTPs are present. If desired, selective repair can be performed by supplying a single dNTP or a selected dNTP within the constraints indicated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with phenol / chloroform and ethanol precipitated. Treatment with S1 nuclease or Bal-31 under appropriate conditions results in hydrolysis of any single stranded portion.
[0041]
The ligation can be performed in a volume of 15-50 μl under the following standard conditions and temperature: 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 mg / ml BSA, 10 mM-50 mM NaCl, and 40 μM ATP, 0.01-0.02 (Weiss) units of T4 DNA ligase at 0 ° C. (ligation of “sticky ends”) or 1 mM ATP, 0.3-0.6 (Weiss ) Either at 14 ° C ("blunt end" ligation) using unit T4 DNA ligase. Intramolecular “sticky ends” are usually ligated at a total DNA concentration of 33-100 μg / ml (total end concentration of 5-100 mM). Intramolecular blunt end ligation is performed at a total end concentration of 1 μM (usually using a 10-30 fold molar excess of linker).
[0042]
Thus, in the present invention, lentiviral packaging vectors were determined by those skilled in the art as involving promoters and gag, pol, rev, env or combinations thereof, and the specific function or actual deletion of tat. Created to include other optional or necessary regulatory sequences and optionally other lentiviral accessory genes.
[0043]
A lentiviral transfer vector (Naldini et al., Supra; Proc. Natl. Acad. Sci., 93: 11382-11388 (1996)) is used to infect human cells with growth-arrested in vitro. And used to transduce nerves after direct injection into adult rat brain. This vector is efficient at the time of marker gene transfer into nerves in vivo, and long-term expression is achieved when there is no detectable pathology. The animals analyzed over the last 10 months after the single injection of the vector, the longest tested so far, showed no reduction in mean levels of transgene expression and no signs of histopathology or immune response ( Blomer et al., J. Virol., 71: 6641-6649 (1997)). Improved versions of lentiviral vectors have been developed in which the HIV virulence genes env, vif, vpr, vpu and nef have been deleted without compromising the ability of the vector to transduce non-dividing cells. Various attenuated versions have shown substantial improvement in the biosafety of the vector (Zufferey et al., Supra).
[0044]
In transduced cells, integrated lentiviral vectors generally have an LTR at each end. The 5 'LTR can cause accumulation of "viral" transcripts that can be recombinant substrates, especially in HIV-infected cells. The 3 'LTR promotes downstream transcription with risks resulting from activating cellular proto-oncogenes.
[0045]
The U3 sequence contains most of the HIV LTR. This U3 region contains enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Some of the promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved in virus isolates and have been shown to be critical virulence factors for virulence. Enhancer elements can act to affect the replication rate in various target cells of the virus (Marthas et al., J. Viol., 67: 6047-6055 (1993)).
[0046]
Since viral transcription begins at the 3 'end of the U3 region of the 5' LTR, these sequences are not part of the viral mRNA and their copies from the 3 'LTR are both in the integrated provirus. Acts as a template for LTR generation. If the 3 'copy of the U3 region is altered in the retroviral vector construct, this vector RNA is still produced from the complete 5' LTR of the producer cell but cannot be regenerated in the target cell. Transduction of such vectors results in inactivation of both LTRs in the progeny virus. Thus, retroviruses are self-inactive (SIN) and these vectors are known as Sin transfer vectors.
[0047]
However, the degree of 3 'LTR deletion is limited. First, the 5 'end of the U3 region provides other essential functions in vector introduction that are necessary for integration (terminal dinucleotide + att sequence). Thus, the terminal dinucleotide and the att sequence can represent the 5 'boundary of the deleted U3 sequence. In addition, some ambiguously defined regions can affect the activity of polyadenylation sites downstream of the R region. Excessive deletion of the 3 'LTR U3 sequence may reduce polyadenylation of vector transcripts with deleterious consequences for both vector titer in producer cells and transgene expression in target cells. it can. On the other hand, limited deletions cannot abrogate LTR transcriptional activity in transduced cells.
[0048]
The new version of the lentiviral transfer vector described herein retains an increasing deletion of the U3 region of the 3 ′ LTR (FIG. 1: U3 from nucleotide -418 to the specified position of the U3 LTR). Deletion length: SIN-78, SIN-45, SIN-36 and SIN-18). Lentiviral vectors have been developed that have an almost complete deletion of the U3 sequence from the 3 'LTR without compromising both the titer of the vector in the producer cell and the expression of the transgene in the target cell. The most extensive deletions (-418 to -18) extend to the TATA box, which results in the inactivation of LTR transcriptional activity in the transduced cells. Therefore, the lower limit for 3 'deletion extends to the position containing the TATA box. This deletion is the remainder of the U3 region up to the R region. This means that the safety of the vector is dramatically increased. Various deletions were made by practices known in the art.
[0049]
Surprisingly, the average expression level of the transgene was higher even in cells transduced with the SIN vector compared to the more complete vector. This is probably due to the removal of transcriptional interference from the upstream HIV LTR on the internal promoter. Such SIN-type vectors with extensive deletions in the U3 region cannot be produced for retroviral vectors based on murine leukemia virus (MLV) without compromising transduction efficiency.
[0050]
The 5 'LTR of the transfer vector was modified by replacing some or all of the transcriptional regulatory elements in the U3 region with a heterologous enhancer / promoter. This change will enhance the expression of the transduced vector RNA in producer cells; to allow the generation of vectors in the absence of the HIV tat gene; and to “rescue” the aforementioned SIN vector It was created to remove a wild type copy of the upstream HIV LTR that could recombine with the 3 ′ deleted version.
[0051]
Therefore, the 5 ′ vector, which is a vector containing the above-mentioned changes in the 5 ′ LTR, can find use as an introduction vector for combination with a sequence that enhances expression and a packaging cell that does not express tat. .
Such 5 ′ vectors can also retain modifications in the 3 ′ LTR, as explained earlier, and can not only be used in packaging cells that do not simply enhance expression and do not express tat. This results in an improved transfer vector that can be further self-inactivated.
[0052]
Transcription from the HIV LTR is highly dependent on the transactivator function of the tat protein. In the presence of a tat often expressed by the core packaging construct present in the producer cell, transcription of the vector from the HIV LTR is strongly stimulated. Since full-length “viral” RNA has complete complementarity of the packaging signal, this RNA is efficiently encapsidated in vector particles and introduced into target cells. The amount of vector RNA available for producer cell packaging is the rate-limiting step in the production of infectious vectors.
[0053]
The enhancer region or enhancer and promoter region of the 5 'LTR was replaced by the human cytomegalovirus (CMV) or mouse rous sarcoma virus (RSV) enhancer or enhancer and promoter, respectively. See Figure 2 for an overview of the construct and hybrid vector code names. CCL and RRL vectors are completely substituted in the 5 'U3 region.
[0054]
A panel of control lentivector HR2 and 5 'hybrids were compared in producer cells transfected with the transfer vector in the presence or absence of packaging constructs providing tat transactivator. The transcription level of the four chimeric vectors is higher than that of the control lenti vector, both in the presence and absence of the packaging construct. All chimeric vectors efficiently introduce the transgene into the target cells, and the RRL vector acts like the control HR2 vector. Finally, integration of the vector into target cells was confirmed by examining cells transduced at early and late passages after transduction. There was no reduction in the ratio of transgene-positive cells indicating that the vector was integrated.
[0055]
The high level expression of the 5 'LTR-modified transfer vector RNA obtained in the producer cells in the absence of the packaging construct indicates that the resulting vector functions in the absence of the functional tat gene. Is shown. The loss of function of the tat gene previously shown for the packaging plasmid described herein results in a higher level of biosafety for Lentiantiviral vector systems conferred by many pathological activities associated with the tat protein. Will. Thus, a lentiviral vector with significantly improved biosafety is a SIN transfer vector that does not have a wild type copy of HIV LTR at either the 5 'end or the 3' end, which is described herein. Used in conjunction with tat-free packaging vectors.
[0056]
Viral supernatant is collected using conventional methods such as filtration of the supernatant 48 hours after transfection. The titer of the virus is 106 NIH 3T3 cells or 10Five It is measured in the presence of 8 μg / ml polybrene (Sigma Chemical Co., St. Louis, MO) by infection of individual HeLa cells. After 48 hours, transduction efficiency is assayed.
[0057]
Thus, the present invention provides methods and means for producing high titer recombinant viruses. Using such a viral particle preparation, target cells can be infected by methods known in the art. Thus, the present invention will find use in ex vivo gene therapy applications where target cells are removed from the host, transformed in the medium using known methods, and then returned to the host.
[0058]
The detailed description of the invention set forth below is a non-limiting example that illustrates various embodiments of the invention.
Example 1
Construction of lentiviral packaging plasmid
The lentiviral packaging plasmid was derived from the plasmid pCMVΔR8.9 (ΔVpr ΔVif ΔVpu ΔNef) previously described in the Zufferey et al. The remaining sequence of the nef gene of pCMVΔR8.9 was removed by digestion with XhoI and BstEII, filled with Klenow and religated. The construct was deleted for 100 base pairs and the HIV-1 truncated env reading frame was ligated to the polyadenylation site of genomic insulin, resulting in plasmid pCMVΔR8.73.
[0059]
In another embodiment of the invention, the plasmid pCMVΔR8.73 is deleted of 133 base pairs of the CMV-derived sequence downstream of the CMV promoter. This sequence contained a splice donor site, which was removed by digestion with SacII of plasmid pCMVΔR8.73 and religation into a larger fragment, resulting in plasmid pCMVΔR8.74.
[0060]
In another embodiment of the invention, all but the consensus 5 'splice donor site remaining in the plasmid pCMVΔR8.74 upstream of the start codon of the gag gene was removed. At the same time, the sequence upstream of the gag gene was changed with optimal translation efficiency to obtain plasmid pCMVΔR8.75. pCMVΔR8.75 was derived from pCMVΔR8.74 and was obtained by replacing the 94 base pair SstII-ClaI fragment with an SstII-ClaI oligonucleotide linker consisting of: 5'-GGGACTGGTGAGTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3 '1) And 5'-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACCCATGGCGGCGGCAGATCTCGAATTCACTCACCAGTCCCGC-3 '(SEQ ID NO:2).
[0061]
In another embodiment of the invention, the inducible packaging construct replaces the PstI-SacII fragment of pCMVΔR8.74 containing the CMV promoter with seven tandem copies of the tetracycline operator sequence linked to the minimal CMV promoter. Obtained. The tet-regulated packaging plasmid pTetΔR8.74 was obtained.
Example 2
Construction of lentiviral transfer vector
The lentiviral transfer vector plasmid was derived from the plasmid pHR'-CMV-LacZ previously described in Naldini et al. (Sci., 272: 263-267 (1996)). pHR2 is such that 124 base pairs of the nef sequence upstream of the 3 'LTR in pHR' are replaced with both polylinkers to reduce the HIV1 sequence and facilitate transgene cloning. Lentiviral transfer vector. pHR2 is derived from pHR'-CMV-LacZ, a 4.6 kb ClaI-StuI fragment was generated by PCR using pHR'-CMV-LacZ as a template and the following as an oligonucleotide with a 828 base pair ClaI- Obtained by replacing the StuI fragment with a 4.4 kb StuI-NcoI fragment and a 4.5 kb NcoI-ClaI fragment derived from pHR'-CMV-LacZ at three sites: 5'-CCATCGATCACGAGACTAGTCCTACGTATCCCCGGGGACGGGATCCGCGGAATTCCGTTTAAGAC-3 '(SEQ ID NO:3) And 5'-TTATAATGTCAAGGCCTCTC-3 '(SEQ ID NO:4).
[0062]
In another embodiment of the invention, pHR3 is a lentiviral vector in which 148 base pairs (including ATG) of the env coding sequence upstream of the Rev response element (RRE) of pHR2 are deleted. pHR3 was derived from pHR2 and was obtained by replacing the 839 base pair NotI-SpeI fragment of pHR2 with a 747 base pair NotI-SpeI fragment obtained by PCR using pHR2 as template and the following as oligonucleotide primers: : 5′-GCGGCCGCAGGAGCTTTGTTCCTTGG-3 ′ (SEQ ID NO:5) And 5'-TACGTAGGACTAGTCTCG-3 '(SEQ ID NO:6).
[0063]
In another embodiment of the invention, pRH5 is a lentiviral transfer vector in which the 310 base pair gag coding sequence (the entire gag coding sequence downstream of the 15th amino acid of the Gag protein) has been deleted from pHR2. pHR5 is digested with NruI of pHR2, NotI linker (synthetic oligonucleotide 5'-TTGCGGCCGCAA-3 ', (SEQ ID NO:7)), Digestion with NotI to excise a 310 base pair fragment, followed by religation.
[0064]
In another embodiment of the invention, pRH6 is a wrench in which the 5 ′ splice donor signal has been mutated (TGGT to TGAT) to enhance the production of full-length transcripts that can be packaged. Viral vector. pHR6 was derived from pHR5 and was obtained by replacing the 239 base pair AflII-ApoI fragment with a 239 base pair AflI-ApoI fragment generated by PCR using pHR2 as template and the following as oligonucleotide primers: 5 '-CCACTGCTTAAGCCT-3' (SEQ ID NO:8) And 5'-CAAAATTTTTGGCGTACTCATCAGTCGCCGCCCCTCG-3 '(SEQ ID NO:9).
[0065]
All PCR fragments were generated by first cloning the PCR reaction product directly into the TA cloning vector pCR2.1 (Invitrogen), followed by sequence verification and excision with the appropriate enzyme.
Example 3
Construction of 5 'LTR chimeric lentivirus vector
In another embodiment of the invention, the 5 'LTR of the lentiviral vector comprises an enhancer and promoter from the Rous sarcoma virus (RSV) U3 region linked to the R region of HIV-1 (plasmid pRRL). pRRL is a lentiviral transfer vector in which an RSV enhancer and promoter (nucleotides -233 to -1 relative to the transcription start position) are accurately fused to the R region of HIV-1 using an oligonucleotide linker. . pRRL refers to WO 97/07225, is derived from plasmids pRT43.RSV.F3 and pRH2, and the 3.4 kb EcoRI-HpaI fragment of pRT43.RSV.F3 is replaced with the 0.67 kb BglII-NotI fragment of pHR2. And a 1.7 kb NotI-StuI fragment of pHR2, 5′-AATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGGGTCTCTCTGGTTAGACCA-3 ′ (SEQ ID NO:10) And 5'-GATCTGGTCTAACCAGAGAGACCCGTTTATTGTATCGAGCTAGGCACTTAAATACAATATCTCTGCAATGCGGC-3 '(SEQ ID NO:11) Was replaced by a synthetic EcoRI-BglII oligonucleotide linker.
[0066]
In another embodiment of the present invention, the 5 ′ LTR of the lentiviral vector comprises Rous sarcoma virus (RSV) bound to the promoter region of HIV-1 (from −78 base pairs relative to the transcription start position). Contains an enhancer (nucleotides -233 to -50 relative to the transcription start position) (plasmid pRLL).
pRLL is a lentiviral transfer vector in which an RSV enhancer is fused to an HIV-1 promoter region using an oligonucleotide linker. pRRL was derived from plasmids pRT43.RSV.F3 and pHR2, and the 3.4 kb EcoRI-HpaI fragment of pRT43.RSV.F3 was replaced with the 0.724 kb AlwNI-NotI fragment of pRH2 and the 1.7 kb NotI-StuI fragment of pRH2. 5′-AATTGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3 ′ (SEQ ID NO:12) And oligonucleotide 5'-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCC-3 '(SEQ ID NO:13) Was replaced by a synthetic EcoRI-AlwNI oligonucleotide linker.
[0067]
In another embodiment of the present invention (plasmid pCCL), the 5 ′ LTR of the lentiviral vector is an immediate early enhancer of human cytomegalovirus (CMV) bound to the R region of HIV-1 and Promoter (Boshart et al. (Cell, 41: 521-530 (1985)) contains the nucleotides -673 to -1 relative to the transcription start position). pCCL is derived from plasmids pRT43.2F3 (US Pat. No. 5,686,279) and pHR2, and the 3.8 kb SstI-HpaI fragment of pRT43.2F3 is replaced with the 1.7 kb BglII-NotI fragment of pRH2 and the 1.7 kb NotI-StuI fragment of pRH2. The
[0068]
In another embodiment of the present invention (plasmid pCLL), the 5 ′ LTR of the lentiviral vector is a cytomegalovirus bound to the promoter region of HIV-1 (from −78 base pairs relative to the transcription start position) ( CMV) contains -220 to -673 enhancer nucleotides relative to the transcription start position. pCLL was derived from plasmids pRT43.2F3 and pHR2, and the 3.6 kb NcoI-HpaI fragment of pRT43.2F3 was replaced with the 0.724 kb AlwNI-NotI fragment of pRH2 and the 1.7 kb NotI-StuI fragment of pRH2, oligo-5'-CATGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGCCC -3 ′ (SEQ ID NO:16) And 5'-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTC-3 '(SEQ ID NO:17By replacement with a synthetic NcoI-AlwNI oligonucleotide linker.
Example 4
Construction of self-inactivating lentiviral vectors
pRRL.SIN-18 was derived from pRRL, and the 400 base pair EcoRV-PvuII fragment in the 3 'LRT was deleted by digestion and religation.
[0069]
pRRL.SIN-36 is derived from pRRL and a 493 base pair BbsI-AlwNI fragment of the 3'-side LTR was synthesized from a synthetic oligonucleotide 5'-GATATGATCAGATC-3 '(SEQ ID NO:18) And 5'-CTGATCA-3 'and a pRRL-derived 0.54 kb AlwN-AvrII fragment and a 6.1 kb AverII-BbsI fragment replaced by an oligonucleotide linker consisting of three sites.
[0070]
pRRL.SIN-45 is derived from pRRL, and the 3 'LTR 493 base pair BbsI-AlwNI fragment was synthesized from the synthetic oligonucleotide 5'-GATATGATCAGAGCCCTCAGATC-3' (SEQ ID NO:19) And 5'-CTGAGGGCTCTGATCA-3 '(SEQ ID NO:20) And a 6.1 kb AlwNI-AvrII fragment and a 6.1 kb AverII-BbsI fragment derived from pRRL.
[0071]
pRRL.SIN-78 is derived from pRRL, and the 493 base pair BbsI-AlwNI fragment of the 3 'LTR is converted into 5'-GATATGATCAGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3' (SEQ ID NO:21) And oligonucleotide 5'-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCCTGATCA-3 '(SEQ ID NO:22) And an oligonucleotide linker consisting of a three-site ligation with a 0.54 kb AlwNI-AvrII fragment and a 6.1 kb AverII-BbsI fragment derived from pRRL.
Example 5
Construction of stable lentiviral packaging cells 00-28 and stable products of lentiviral vectors
Stable lentiviral packaging cells were generated using the 293G cell line. 293G cells were tet from the MD cassette (CMV promoter and intervention sequence from human β globin gene-
[0072]
Four additional p24 / VSV.G positive clones were analyzed for their ability to package lentiviral transfer vectors. These clones were cloned into a transiently generated lentiviral vector (VSV.G pseudotype) containing an A. Victoria green fluorescent protein (GFP) expression cassette activated by the CMV promoter with a multiplicity of infection of 10 and Infection was performed in the presence of polybrene (8 μg / ml). Infected clones were then expanded to remove tetracycline. At 72 hours after induction, the media was collected for 24 hours and the supernatant was filtered and snap frozen. Frozen supernatants were titrated on native HeLa cells for GFP gene transduction. By FACS analysis, the cell population generated by infection of packaging clone 00-28 (referred to as 10-28) has a maximum titer of 5 × 10Four It was determined to have transducing units (T.U.) / Ml.
[0073]
Infected packaging population 10-28 was used to generate high titer producing clones of GFP lentiviral vector. 10-28 cells were examined by FACS and retained and expanded with the highest GFP expressing cells. This population was then infected four more times in succession ("ping") with transiently generated GFP lentivirus (VSV.G pseudotype). After each infection, supernatants were collected 72-96 hours after VSV.G induction. The supernatants were titered on HeLa cells and analyzed for p24 content by immunocapture assay. The infectious titer peaked after the third ping, 1.5 x 106T.U./ml was reached (see FIG. 3). The cell population from the third ping was subcloned and the producer of the high titer vector was isolated.
[0074]
All articles and patent applications cited herein are hereby incorporated by reference in their entirety so that individual articles or patent applications are clearly and individually indicated to be incorporated by reference.
As will be apparent to those skilled in the art to which the present invention pertains, the present invention also encompasses transfection and transduction in forms other than those specifically shown above, such as transfection and transduction of other mammalian cell types. Or as long as they do not depart from the essential features. Accordingly, the specific embodiments of the present invention as described above are to be regarded as illustrative and not restrictive. The scope of the invention is, as noted, not limited to the embodiments limited in the foregoing description, but is the appended claims.
[Brief description of the drawings]
1 shows various lentiviral vectors. RSV is the Rous sarcoma virus enhancer / promoter; R is the R region of the LTR; U5 is the U5 region of the LTR; SD is the splice donor site, eg, the
FIG. 2 shows another lentiviral vector. CMV is a cytomegalovirus. In other respects, the symbols are the same as those shown in FIG.
FIG. 3 is a graph showing graded vector production with increasing amounts of transfer vector.
[Sequence Listing]
Claims (23)
a)i)請求項7記載の少なくとも1種のレンチウイルスパッケージングプラスミド;及び
ii)前記レンチウイルスに対して内因性でない発現されたenv遺伝子を保持する前記レンチウイルスに対し内因性でない発現プラスミド;
により細胞を形質転換し、パッケージング細胞を得る工程;
b)前記パッケージング細胞を、発現された異種遺伝子を保持するレンチウイルス導入ベクターにより複数の形質転換を行い、プロデューサー細胞を得る工程;
c)前記プロデューサー細胞を培地において培養する工程;及び
d)前記プロデューサー細胞を前記培地から分離し、前記組換えレンチウイルスベクターを前記培地から回収する工程。A method for producing a recombinant lentiviral vector comprising the following steps:
a) i) at least one lentivirus packaging plus Mi de of claim 7; and
ii) non-endogenous to said lentivirus which holds the env gene is expressed not endogenous to the lentivirus expression plasmid;
Transforming the cells to obtain packaging cells;
b) performing a plurality of transformations of the packaging cell with a lentiviral transfer vector carrying the expressed heterologous gene to obtain a producer cell;
c) culturing the producer cell in a medium; and
d) separating the producer cell from said medium, recovering said recombinant lentiviral vector from said medium.
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US08/989,394 US5994136A (en) | 1997-12-12 | 1997-12-12 | Method and means for producing high titer, safe, recombinant lentivirus vectors |
PCT/US1998/025719 WO1999031251A1 (en) | 1997-12-12 | 1998-12-11 | Method and means for producing high titer, safe, recombinant lentivirus vectors |
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EP (1) | EP1036182B1 (en) |
JP (2) | JP4640742B2 (en) |
KR (1) | KR20010033064A (en) |
AU (1) | AU751985B2 (en) |
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